Abstract: The long-term objective of the proposed work is the elucidation of the mechanism of DNA replication in animal mitochondria, and its relationship to mitochondrial mutagenesis and human disease. A combined approach of current methods in biochemistry, structural biology and molecular genetics will be pursued to study the mechanism, structure and physiology of the mitochondrial replisome, with a focus on the replicative DNA helicase and the mitochondrial single-stranded DNA-binding protein. In combination with the extensive studies by us and others on the key replicative enzyme in mitochondria, DNA polymerase 3, we expand the scope of prior research to study the assembly and function of proteins at the mitochondrial DNA replication fork, and to probe the modes of mitochondrial DNA replication in vivo and in vitro. Mitochondria are the energy-producing organelle in animals and mitochondrial function impacts nearly every aspect of cellular function. Thus, mitochondria play a major role in human health and likewise, mitochondrial dysfunction is intricately associated with human disease. Mitochondrial dysfunction is implicated in a wide range of neurological diseases, in metabolic diseases, in muscular dystrophies and nephropathies, and in a broad spectrum of named disorders. Defects in mitochondrial biogenesis lead to mitochondrial DNA mutation, depletion and deletion syndromes that result in loss of mitochondrial and subsequent cellular function. The prevalence of mitochondrial genetic disease and recent recognition of the mitochondrial toxicity of antiviral and antimicrobial drugs the critical need for an in-depth understanding of the structure and functions of the mitochondrial DNA replication apparatus. PUBLIC HEALTH RELEVANCE: Narrative: A common feature in diverse chronic, age-related diseases is a breakdown or disturbance in the cell's ability to produce energy. Central to the cellular energy production is the mitochondrion, an organelle that plays a crucial role in multiple metabolic processes, and whose dysfunction is linked directly to many pathologies, including those most represented in an aging population. Therefore, a clear understanding of mitochondrial biogenesis and energy production and how these processes are deregulated in common disease states, is essential to treating them effectively (and economically).